PDLIM5在神经元生长锥生长中的作用及其机制

Growth and development of neurite is one of major aspects in nervous repair, regeneration and tissue engineering. The outgrowth and collapse of the growth cone plays critical role in the developing of neurite. However, little is known on the regulation mechanism of this key event in neuron. Our previous studies revealed that PKCε participates in the collapse of growth cone, and interference of PKCε with specific shRNA inhibits PMA induced hippocampal neuron growth cone collapse. Our further studies have indicated that PDLIM5 can bind PKCε specifically and plays as a cargo in the translocation of PKCε. Other research data showed that PDLIM5 could interacts with several cytoskeleton proteins, such as actin, in cardiomyocytes and skeletal muscle cells. Therefore, we assume a hypothesis that PDLIM5 and the PDLIM5-PKCε complex participate in the regulation of neural growth cone. We intend to use the shRNA interference, point mutation, pulldown assay,immunostaining and calcium imaging approaches, to prove that PDLIM5 participates in the collapse of the growth cone and identify the effect of PDLIM5 and PKCε on the regulation of the neurite outgrowth. The study results may reveal the mechanisms of neural growth and development mediated by the PDLIM5 and provides some candidate targets for nervous tissue engineering... .Keywords：PDLIM5, growth cone, scaffold protein, PKCε, signal transduction

神经元突起生长发育是神经修复、再生及神经组织工程学的重要研究内容，而生长锥的生长与坍塌又是神经突起发育的关键事件。然而对该事件的细胞内调节机制知之甚少。我们前期研究证实，PKCε参与了生长锥的坍塌过程，shRNA 干扰大鼠海马神经元PKCε后能抑制生长锥坍塌；此外PDLIM5蛋白与PKCε呈钙离子浓度依赖性结合，帮助PKCε转位。有研究表明PDLIM5在心肌/骨骼肌细胞内可与actin骨架蛋白相互作用，从而参与了细胞生长与运动的调控。因此我们提出PDLIM5及PDLIM5-PKCε复合物可能调控生长锥发育的科学假说。本项目拟采用小分子干扰、点突变、pulldown、荧光影像及钙成像等技术，获得PDLIM5参与生长锥坍塌过程的直接证据，并阐明PDLIM5及PDLIM5-PKCε复合物在调节生长锥及神经元突起生长中的作用。为揭示神经元生长发育过程以及寻找神经组织再生的新靶标提供可靠实验依据。

神经元生长锥的生长与坍塌是神经突起发育的关键事件，然而对该事件的细胞内调节机制知之甚少。我们前期研究证实，PKCε参与了生长锥的坍塌过程，shRNA 干扰大鼠海马神经元PKCε后能抑制生长锥坍塌；此外PDLIM5 蛋白在神经元中可与PKCε结合，帮助PKCε转位。因此我们提出PDLIM5可能具有调控生长锥发育功能的科学假说。本项目采用小分子干扰、蛋白质相互作用、免疫荧光及梯度离心等技术，得出以下结论：.1.PDLIM5可分布在神经元生长锥中，并与PKCε具有共定位。PDLIM5在生长锥中的主要可与α-actinin及PKCε结合，而不与actin或tubulin结合。.2.抑制PDLIM5的表达，可保护神经元不受由PMA刺激所导致的生长锥坍塌。该现象与抑制PKCε的表达或活性对神经元生长锥的作用相似。.3.PDLIM5可与PKCε在生长锥相互作用并使之发生转位，从而介导了PKCε的激活对神经元生长锥的坍塌作用。.以上结果为证实PDLIM5 参与生长锥发育调控的科学假说提供了直接证据，这为进一步揭示PDLIM5在神经元生长发育中的作用奠定了实验基础。